When grown in the presence of trace amounts of selenium, Escherichia coli synthesize lysine and glutamate accepting species of tRNA which contain the selenonucleoside, 5-methylaminomethyl-2-selenuridine (mnm5se2U). The selenonucleoside was quantitated in bulk, hydrolyzed tRNA by monitoring at 313 nm during reversed phase high performance liquid chromatography. E. coli tRNA from cells grown in rich, natural media such as beef extract or tryptone-yeast extract, without intentional selenium supplementation, contained 4 to 61 pmol mnm5se2U per A260 unit of tRNA. Thus, there is probably enough selenium present in natural environments for mnm5se2U synthesis to occur. In tRNA from cells grown on selenium-supplemented media (1 MuM selenite or selenate) a maximum of 60-70 pmol mnm5se2U per A260 unit was observed, or about 4 mol mnm5se2U per 100 mol tRNA. Under these conditions an approximately equal amount of 5-methylaminomethyl-2-thiouridine (mnm5s2U) was also present. The amount of mnm5se2U did not increase and the amount of mnm5s2U did not decrease from these values when the culture medium selenium was increased (or if sulfur was decreased) to levels which were inhibitory to growth. A mutant defective in the synthesis of mnm5s2U (obtained from M. G. Marinus) incorporated the same amount of selenium into tRNA as the parent strain. The selenonucleoside in the mutant was identified as 5-aminomethyl-2-selenouridine. Studies with wild-type cells, employing chloramphenicol and rifampicin, suggested that a stable tRNA species existed in selenium-deprived E. coli which could be modified with selenium to form mnm5se2U-containing tRNA. By labeling mnm5s2U residues with S-35 it was demonstrated that synthesis of mnm5se2U was accompanied by a loss of sulfur from mnm5s2U residues. Finally, it was shown that purified E. coli tRNA-Lys or rRNA-Glu, containing mnm5s2U, could serve as s substrate in a crude, in vitro, selenium incorporation system. Synthesis of mnm5se2U in this system was completely dependent upon addition of ATP. This is the first observation, in vitro, of a specific enzymatic incorporation of selenium into a macromolecule.